The overall objective of the project is to identify the sweet taste receptor recognition sites and taste modification sites of two intensely sweet proteins, monellin and thaumatin. These two are about 100,000 times sweeter than sugar on a molar basis, and are the most potently sweet molecules known to man. During the previous granting period we determined the crystal structures of both proteins at 3.0 Angstroms resolution. We propose to determine the receptor recognition sites and taste modification determinant sites by a combination of biochemical, cloning, and x-ray crystallographic methods. The starting point of the proposal is to clone the genes for the two proteins and express them in E. coli or yeast. Once such clones are established it is relatively easy to generate mutant proteins. The choice of mutants to be made will be based on the crystal structures of the two proteins. We have already cloned and expressed the "fused" monellin gene and are in teh process of completing the same for thaumatin. Three of the monellin mutants have so far been crystallized.